PROJECT SUMMARY A significant limitation in natural product drug discovery is the inability to effectively convert sequence information to new compounds in a rapid and high throughput fashion. The long-term objective of this project is to systematically address this issue with the goal of re-establishing the once vibrant pipeline of environmentally-derived natural compounds, with a special emphasis on discovering new antibiotics. The final vision is the development of a microfluidics biosynthetic platform capable of generality, speed, throughput, and economy in natural product pathway reconstitution and compound discovery. The goal of the current R21 application is captured in the following specific aim: To generate the complex natural product antibiotic erythromycin A using cell-free biosynthesis. By doing so, precedent will be set for the ability to produce such compounds using a purely in vitro approach. This and additional preliminary data collected through the proposed work will then be the basis for continual research towards a cell-free biosynthetic platform capable of precise engineering and ultra-high throughput. As a result, the platform will allow unprecedented access to the broadest range of new chemical entities, including novel antibiotic compounds, while being unencumbered by the constraints of a cellular host.